We propose to study the structure, chromosomal organization and molecular genetics of the Drosophila alcohol dehydrogenase (Adh) gene using molecular cloning procedures and classical genetic techniques. Our primary experimental approach will be the analysis of differences in gene structure and expression between wild-type flies and flies that are mutant in Adh gene expression. Large numbers of Adh mutants will be generated using X-irradiation or chemical mutagens in conjunction with an ethanol selection screen for flies lacking Adh activity. Mutants that display aberrant Adh mRNA synthesis will be classified according to the nature of their defects by biochemical experiments (cDNA and RNA blotting procedures solution hybridization experiments and nucleotide sequence analysis) and classical genetic techniques (recombination mapping, complementation and reversion studies). We also plan to make use of biochemical and genetic techniques to investigate the possible relationship between chromatin structure and gene activation. This will be accomplished by isolating and characterizing a large region of chromosome 2 which contains the Adh gene (200 kilobases) and then using this information to guide nuclease digestion experiments which will be carried out with nuclei obtained from normal and mutant flies. In order to identify sequences which are necessary for Adh gene transcription and mRNA processing we plan to develop in vivo and in vitro assays for Adh gene expression. In vivo expression systems will include microinjection of Drosophila embryos with cloned Adh genes and the propagation of SV40 DNA-Adh gene hybrids in mammalian cells in culture. An in vitro transcription assay which involves the use of whole cell extracts will also be employed. The use of these procedures should make it possible to identify sequences which are involved in Adh gene transcription and Adh mRNA processing and translation. In order to identify sequences which are involved in differential gene expression in development, we will attempt to establish an embryo transformation system for Drosophila using cloned Adh DNA. The behavior of wild-type and mutant Adh genes will be compared in each of these assays of gene expression.